Rapid measurements of fluorescence from individual cells by flow cytometry allows large samples of cells, organisms and subcellular components to be statistically analyzed in a short period of time. See, e.g., Flow Cytometry and Sorting, 2.sup.nd edition, M. R. Melamed, T. Lindmo, and M. L. Mendelsohn eds., Wiley-Liss, N.Y., 1990. For flow cytometric analysis, the cells are stained with fluorescent probes that bind to a specific subcellular component. The fluorescence intensity from these probes, upon excitation by a continuous wave light source, gives a measure of the particular subcellular component in the cell. Flow cytometric measurements find application in a wide range of biological areas such as cell biology (See, e.g., "Cytochemical Techniques For Multivariate Analysis Of DNA And Other Cellular Constituents," by H. A. Crissman and J. A. Steinkamp, in Flow Cytometry And Sorting, 2.sup.nd edition, M. R. Melamed, T. Lindmo, and M. L. Mendelsohn eds., Wiley-Liss, N.Y., 1990, pages 227-248, and "Quantitative Cell Cycle Analysis," by J. W. Gray, F. Dolbeare, and M. G. Pallavicini, in Flow Cytometrv And Sorting, 2.sup.nd edition, M. R. Melamed, T. Lindmo, and M. L. Mendelsohn eds., Wiley-Liss, N. Y., 1990, pages 44514 468.), chromosome analysis and sorting (See, e.g., "Flow Karyotyping And Chromosome Sorting," by J. W. Gray and L. S. Cram in Flow Cytometry And Sorting, 2.sup.nd edition, M. R. Melamed, T. Lindmo, and M. L. Mendelsohn eds., Wiley-Liss, N.Y., 1990, pages 50314 530.), immunology (See, e.g., "Immunofluorescence Techniques," by M. R. Loken, in Flow Cvtometry And Sorting, 2.sup.nd edition, M. R. Melamed, T. Lindmo, and M. L. Mendelsohn eds., Wiley-Liss, N.Y., 1990, pages 341-353.), hematology (See, e.g., "Analysis And Sorting Of Blood And Bone Marrow Cells," by J. W. M. Visser in Flow Cytometry And Sorting, 2.sup.nd edition, M. R. Melamed, T. Lindmo, and M. L. Mendelsohn eds., Wiley-Liss, N.Y., 1990, pages 669-684.), and microbiology (See, e.g., "Application Of Flow Cytometry On Bacteria: Cell Cycle Kinetic, Drug Effects And Quantitation Of Antibody Binding," by H. B. Steen et al., Cytometry 2, 249-257 (1982).).
Recent advances in fluorescence lifetime spectroscopy have shown that the study of fluorescence decay and excited state lifetimes can provide valuable information on subcellular organization, the fluorescence decay of the probes being sensitive to the surrounding microenvironment. See, e.g., Principles Of Fluorescence Spectroscopy, by J. R. Lakowicz, Plenum Press, New York, 1983, and Mechanisms Of Energy Transfer: In Biophysics, W. Hoppe, W. Lohmann, H. Markl, and H. Ziegler eds., Springer Verlag, Berlin, 1983. Fluorescence decay can provide insight into intermolecular interactions at the subcellular level, which are often accompanied by changes in the fluorescence decay of the molecules involved. See, e.g., "Mechanism of Fluorescence Concentration Quenching of Carboxyfluorescein in Liposomes-Energy Transfer to Non-Fluorescent Dimers," by R. F. Chen et al., Anal. Biochem. 172, 61-77 (1988). As a result, it is important to know if any change in the characteristic of the fluorescence decay or the fluorescence lifetime has occurred in a system under investigation. A flow cytometric method that permits detailed time-resolved analysis of fluorescence decays in single cells, is not available. Such analysis by flow cytometry is expected to have many advantages over similar measurements in bulk solution or suspensions in a cuvette (See, e.g., "Fluorescence Lifetime Analysis of DNA Intercalated Ethidium Bromide and Quenching by Free Dye," by D. P. Heller et al., Biophys. Chem. 50, 305-312 (1994) and "Analysis of Fluorescence Quenching in Some Antioxidants from Nonlinear Stern-Volmer Plots," by H. Zeng et al., J. Luminescence 63, 75-84 (1995)), which is another method by which fluorescence decay studies in biological samples are conducted. For example, experiments in a cuvette provide only an average measure for the fluorescence properties of the collection of all the cells contained in the sample. See e.g., D. P. Heller, supra, and "Nanosecond Fluorescence Microscopy," by S. M. Keating et al., Biophys. J. 59, 186-202 (1991). Additionally, since the illumination volume in such measurements is relatively large, the interference from the background signals (fluorescence from unbound dye, Raman scattering from the medium of suspension, etc.) can be significant. A microscope-based system permits analysis of individual cells on a slide. See, S. M. Keating, supra, and "Development of a Streak-Camera Based Time-Resolved Microscope Fluorimeter and Its Application to Studies of Membrane Fusion in Single Cells," by A. Kusumi et al., Biochem. 30, 6517-6527 (1991). However, the speed at which the cells can be studied is slow because each cell must be brought into the illumination region manually. By contrast, however, in a flow cytometer not only is each cell sampled and measured individually, but the rate of sampling is high. Hundreds of cells can be measured per second in a flow cytometer. Since the cells pass through the laser beam in single file, no further adjustments are required from cell to cell, once the flow cytometer is properly aligned. See, e.g., "Flow Cytometer for Resolving Signals From Heterogeneous Fluorescence Emissions and Quantifying Lifetime in Fluorochrome Labeled Cells and Particles by Phase-Sensitive Detection," Rev. Sci. Instrum. 64, 3440-3450 (1993), and "Fluorescence Lifetime Measurements in a Flow Cytometer by Amplitude Demodulation Using Digital Data Acquisition Technique," by C. Deka et al., Cytometry 17, 94-101, (1994). Further, for the fluorescence decay measurements using a microscope, a synchronously pumped cavity dumped dye laser was used (S. M. Keating, supra), with the pump laser being a mode-locked argon-ion laser. By contrast, laser-based flow cytometers use cw lasers for most conventional applications. See, e.g., Melamed, Crissman, Gray, Loken, Visser, and Steen, supra. By using an electrooptic modulator (EOM) it should be possible to conveniently adapt a cw laser for pulsed time-resolved fluorescence decay measurements instead of using an expensive and complicated multilaser, cavity-dumped system.
Measurements of apparent fluorescence lifetimes in single cells have been made in flow cytometers by frequency domain spectroscopy (See, e.g., Lakowicz, supra, and "Phase-Sensitive Fluorescence Spectroscopy: A New Method to Resolve Fluorescence Lifetimes and Emission Spectra of Components in a Mixture of Fluorophores," J. R. Lakowicz et al., J. Biochem. Biophys. Methods. 5, 19-35 (1981)) using a cw laser sinusoidally modulated at a single modulation frequency. See, also., Lakowicz, supra, and "Phase-Resolved Fluorescence Lifetime Measurements for Flow Cytometry," P. G. Pinsky et al., Cytometry 14, 123-135 (1993). In this method, an apparent lifetime is measured from the phase-shift of the fluorescence signal relative to the modulated excitation at the given modulation frequency. This apparent lifetime gives a true measure of the intrinsic fluorescence lifetime if the fluorescence decay is exponential. A phase shift measured at a single modulation frequency, however, cannot, by itself, determine if a particular decay is exponential in form. Moreover, the lifetimes calculated from the measured phase shifts for multiexponential or nonexponential decays depend on the modulation frequency. See, e.g., J. R. Lakowicz et al., "Picosecond Resolution of Tryosime Fluorescence and Anisotropy Decays by 2 GHz Frequency-Domain Fluorometry," by J. R. Lakowicz et al., Biochem. 26, 82-90 (1987), and "Measuring Fluorescence Decay Times by Phase-Shift and Modulation Using the High Harmonic Content of Pulsed Light Sources," by E. Gratton et al., Nuovo Cimento B15, 110-124 (1980). As a result, a single frequency measurement is not sufficient for the analysis of fluorescence decays that do not follow a single exponential law. It is known that in a system where intermolecular interactions between neighboring fluorochromes lead to nonradiative energy transfer and energy migration, the fluorescence decay becomes nonexponential. See, e.g., J. R. Lakowicz, Dorr, and Chen, supra. In addition, nonexponential decays in flow cytometry can also result simply from the fact that the cells are often labeled with multiple probes having different lifetimes. Therefore, an alternative method is required that permits detailed analysis of arbitrary fluorescence decays from individual cells and particles in flow. Time-domain lifetime spectroscopy offers a convenient solution to this problem. In this situation, if the fluorescence intensity due to a pulsed excitation is measured as a function of time and the system's response function is deconvoluted from the recorded fluorescence data, the impulse response function of the fluorescence decay can be extracted for any arbitrary decay law. See, e.g., J. R. Lakowicz; "Deconvolution of Fluorescence and Phosphorescence Decay Curves-Least Squares Method," by W. R. Ware et al., J. Phys. Chem. 77, 2038-2048 (1973); "Correction of Instrumental Time Response Variation with Wavelength in Fluorescence Lifetime Determinations in the Ultraviolet Region," by D. M. Reyner et al., Rev. Sci. Instrum. 48, 1050-1054 (1977); "On the Analysis of Fluorescence Decay Kinetics by the Method of Least Squares," by A. Grinvald et al., Anal. Biochem. 59, 583 (1974); and "Data Reduction and Error Analysis for the Physical Sciences," by P. R. Bevington, McGraw-Hill, New York, 1969. Time-resolved measurements have been reported for flowing streams of dilute solutions of dye molecules. See, e.g., "Detection and Lifetime Measurement of Single Molecules in Flowing Sample Streams by Laser-Induced Fluorescence," by C. W. Wilkerson et al., App. Phys. Lett. 62(17), 2030-2032 (1993) and "Error Analysis of Simple Algorithms for Determining Fluorescence Lifetimes of Ultradilute Dye Solutions," by S. A. Soper et al., AppI. Spectro. 48, 400-405 (1994). These measurements were made by the gated single-photon counting technique using mode-locked lasers at high repetition rates (76 MHz -82 MHz, pulse-to-pulse period 13.2 ns-12.2 ns), and have been concerned with fluorescence decays having lifetimes of the order of 4 ns or less. The analyses were based on the assumption of a single exponential decay. Due to the high repetition rate of the laser in such systems, however, one could not analyze fluorescence decays having lifetimes longer than one third of the repetition time period of the laser pulses, particularly, if the decay were nonexponential. Further, the duration of the data acquisition for these measurements ranged from 3 ms (Wilkerson, supra) to 10 s (Soper, supra). For flow cytometric analysis of biological cells, however, the data for each cell must be acquired within its transit time across the laser beam. Typically, this is on the order of 20 .mu.s. See, e.g., Steinkamp and Deka, supra. Further, in order to be of practical use for biological studies, the measurement and analysis method must be general enough to include both exponential and nonexponential decays. Finally, it must permit analysis of fluorescence decays having lifetimes up to as long as 25 ns (Heller, supra) or longer. Moreover, since the conventional laser-based flow cytometers already use a cw laser for excitation, it would be advantageous and less expensive if the lifetime sensing method could be adapted to use a cw laser.
In "Fluorescence Detection And Size Measurement Of Single DNA Molecules," by Alonso Castro et al., Analytical Chemistry 65, 849-852 (1993), fluorescence in flowing molecules is detected. However, a time-gate window is employed so that only delayed fluorescence is detected. For investigation of nonexponential decay, it is necessary to observe the characteristics of the fluorescence early in the decay curve. In "Time-Resolved Flow Cytometer For The Measurement Of Lanthanide Chelate Fluorescence: I. Concept And Theoretical Evaluation," by Marc A. Condrau et al., Cytometry 16, 187-194 (1994) and in "Time-Resolved Flow Cytometer For The Measurement Of Lanthanide Chelate Fluorescence: II. Instrument Design And Experimental Results," by Marc A. Condrau et al., Cytometry 16, 195-205 (1994), the authors also discuss the measurement of delayed luminescence.
Accordingly, it is an object of the present invention to perform time-resolved fluorescence decay measurements on single particles in flow.
Another object of the invention is to perform time-domain measurements of the fluorescence decay of fluorochrome-labeled single flowing particles that can be adapted to a conventional flow cytometer (using a cw laser), can readily accommodate a wide range of lifetimes, and can be applied to both exponential and nonexponential decays.
Yet another object of the present invention is to perform time-resolved fluorescence decay measurements on single particles in flow in order to extract the complete decay law governing the fluorochromes in the particles.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.